Date Published: August 01, 2018
Publisher: International Union of Crystallography
Author(s): Dorothee Liebschner, Pavel V. Afonine, Nigel W. Moriarty, Paul Langan, Paul D. Adams.
Models of crystal structures determined by neutron diffraction and deposited in the Protein Data Bank to date were analysed. The lessons learned from this data-mining effort are summarized and suggestions for improvements to the deposition and validation of neutron models are outlined.
The predominant method to determine the three-dimensional structure of macromolecules is X-ray crystallography (Fig. 1 ▸), which is based on the interaction between X-rays and the electrons of the atoms constituting the crystal. Neutron diffraction is a complementary technique that relies on the interaction of neutrons with atomic nuclei. The neutron scattering cross-section, which determines the probability of a neutron being scattered by a nucleus, varies by element (or isotope) in a nonlinear fashion, as opposed to X-rays, where the scattering increases with the number of electrons. This is why neutron diffraction complements X-ray diffraction by enabling the location of very light atoms or ions such as hydrogen or protons in protein structures. As the knowledge of H-atom positions is important for determining the protonation states and reaction pathways of proteins (Engler et al., 2003 ▸; Weber et al., 2013 ▸; Haupt et al., 2014 ▸; Casadei et al., 2014 ▸; Howard et al., 2016 ▸), neutron diffraction is able to provide valuable information for the understanding of catalytic mechanisms and ligand binding (Yamaguchi et al., 2009 ▸; Bryan et al., 2013 ▸; Knihtila et al., 2015 ▸).
Table 2 ▸ provides a summary of the following parameters for all neutron models: PDB code, deposition year, H/D state, refinement program, high-resolution and σ cutoff, published and recomputed Rwork and Rfree. Table 3 ▸ lists the same information for models from joint XN refinement, along with relevant cutoffs and R factors for the X-ray data sets.
The work described in this report led to the development of a new tool in PHENIX that can comprehensively validate neutron models and data. It is available in PHENIX release 1.13 and later. The following validation tasks are performed.(i) Identification of missing H (or D) atoms.(ii) An accounting of the number of H, D and exchanged H/D sites.(iii) Identification of H/D sites with an occupancy ratio that leads to nearly full cancellation of their density (approximately 0.35/0.65). If such a site has a degree of freedom, it should be checked.(iv) Identification of H/D sites with different coordinates, ADPs and unlikely occupancy values.(v) A count of water molecules with zero, one or two D atoms.(vi) A warning message if X-ray X—H distances are used.Broader use of this tool will help address some of the issues that are raised in our analysis.
Neutron models constitute a small fraction of the models deposited in the PDB; however, the information that they provide is unique and of great importance for understanding biological function. At present, X-ray crystallography is the method of choice for determining the structure of biological macromolecules. Neutron crystallography is used only in cases where a critical science question requires the direct localization and visualization of H atoms or protons. The initial goal of surveying neutron models was to verify the suitability of their use in the development and benchmarking of new robust computational tools for neutron crystallography. However, a preliminary assessment of model-to-data fit quality has revealed opportunities to improve the PDB annotation and validation methods and the deposition process itself. Implementation of the suggested improvements will minimize inconsistencies between the deposited neutron models available in the PDB and therefore the possibility of misinterpretation. Most of the issues identified concerned the handling of H and D atoms. The survey led to the development of a new tool in PHENIX that can comprehensively validate H and D atoms in protein models. Since the primary use of macromolecular crystallography is to locate and directly visualize H atoms, it is important to address these issues, so that deposited neutron models allow the retrieval of the maximum amount of information with the smallest effort of manual intervention.